Microbes for Legume Improvement || Growth Promotion of Legumes by Inoculation of Rhizosphere Bacteria

Chapter 9

Growth Promotion of Legumes by Inoculation

of Rhizosphere Bacteria

Satyavir S. Sindhu, Seema Dua, M.K. Verma, and Aakanksha Khandelwal

Abstract Most plants grown in fields are colonized by diverse groups of rhizo-

sphere bacteria that form beneficial or pathogenic relationships with their hosts.

The root exudates encourage the development of beneficial bacterial communities

in the root zone capable of producing secondary metabolites that improve plant

growth and crop yield. These beneficial associations facilitate plant growth either

by enhancing crop nutrition, releasing plant growth stimulating hormones, reducing

damages caused by pathogens/pests by producing antibiotics, bacteriocins, side-

rophores, hydrolytic enzymes and other secondary metabolites or by improving

resistance to environmental pollutants. Rhizosphere bacteria also supply biologi-

cally fixed nitrogen, solubilize bound phosphorus and may provide other nutri-

ents, such as, potassium, iron and sulfur to plants. These beneficial associations

hence, reduce the requirement of chemical fertilizers used for crop productivity.

Moreover, some rhizobacteria are used to relieve the toxicity of metals and

organic toxicants, either through stimulation of microbial degradation of pollu-

tants in the rhizosphere, or by uptake of pollutants/toxicants by the plant. The

inoculation of the legumes with such rhizosphere bacteria has often been found

to increase symbiotic properties, plant biomass and yields under green house or

field conditions. Tremendous progress has been made recently in characterizing

the process of rhizosphere colonization, identification and cloning of bacterial

genes involved in nitrogen fixation, phosphorus solubilization, production of

plant growth regulators and in suppression of plant diseases. The interactions/

relationships of rhizosphere bacteria with their hosts and performance of wild-

type and genetically manipulated beneficial bacterial populations are discussed

for their efficient utilization in legume production under sustainable agriculture

systems.

S.S. Sindhu (*), S. Dua, M.K. Verma, and A. Khandelwal

Department of Microbiology, CCS Haryana Agricultural University, Hisar 125004, India

e-mail: [email protected]

M.S. Khan et al. (eds.), Microbes for Legume Improvement,DOI 10.1007/978-3-211-99753-6_9, # Springer-Verlag/Wien 2010

195

9.1 Introduction

The rhizosphere around the growing plant roots is a very dynamic environment and

harbors a large number of total microorganisms, especially bacteria, greater than

root-free soil. The heterogenous microbial populations interact with each other and

with the plant through symbiotic, associative, neutralist or antagonistic effects. The

outcome of colonization and penetration of the plant tissue with a microorganism

varies from asymptomatic to disease and from associative to symbiosis, depending

upon the mutual perception or recognition between the interacting cells. Such

interactions are influenced greatly by the environment. The microbes that penetrate

and colonize plants have evolved an elaborate system for subverting the plant

defense system. The group of beneficial, root associative bacteria that stimulate

the growth of a plant is termed as plant growth-promoting rhizobacteria (PGPR).

Fluorescent pseudomonads and bacilli comprise the major group among PGPR

along with other bacteria, like, Acetobacter, Agrobacterium, Alcaligenes, Arthro-bacter, Azospirillum, Azotobacter, Cellulomonas, Clostridium, Enterobacter,Erwinia, Flavobacterium, Pasteuria, Serratia and Xanthomonas. The beneficial

rhizosphere microorganisms also include rhizobia and bradyrhizobia, which estab-

lish symbiotic relationship with leguminous plants. In the absence of appropriate

microbial populations in the rhizosphere, plant growth may be impaired (Sturz

et al. 2000).

Legumes are widely used for food, fodder, fuel, timber, green manure, and as

cover crops in different agricultural systems. In developing countries, legumes are

often an integral part of forest, pastures and agricultural ecosystems. On global scale,

nitrogen-fixing legumes are the major source of soil N pool. Legume crops meet their

N requirement through symbiotic N2-fixation by forming nodules with rhizobia.

Legumes and the rhizosphere provide most of the nutritional requirements of nodule

bacteria and enhances the Rhizobium population several folds during plant growth.

Rhizobium–legume associations are usually host specific, and a given rhizobial strain

can infect only a limited number of hosts. Most of the characterized rhizobial strains

have been isolated from the limited range of cultivated legume species.

The high-input agricultural practices of the more industrialized nations of

temperate zones are rarely suitable for tropical conditions in most developing

countries. Therefore, emphasis on biological processes which are able to improve

agricultural productivity, while minimizing soil loss and ameliorating adverse

edaphic conditions, are essential. A better understanding of rhizobial ecology,

optimization of N2-fixing conditions in legume-Rhizobium symbiosis and selection

of rhizosphere bacteria having synergistic interactions with Rhizobium leading to

growth-promoting effects on legumes are crucial for improving and sustaining

agricultural ecosystems. The inoculation effects of diverse bacterial groups posses-

sing plant growth-promoting traits on the performance of legumes are discussed.

The fundamentals of the different processes involved in plant growth promotion are

briefly introduced. The different strategies or biotechnological approaches adopted

for enhancing biological N2-fixation (BNF), P-solubilization, auxins production

196 S.S. Sindhu et al.

and improving biocontrol activity are also described. Various constraints involved

in crop improvement following inoculation with genetically engineered bacterial

strains and the possibilities of deriving desired benefits by ensuring the establish-

ment and survival of introduced microbial inoculants in soil are explored.

9.2 Mechanisms Involved in Plant Growth Promotion

Microbial ecology of the rhizosphere includes the study of the interactions of

microorganisms with each other and the environment surrounding the plant root

(Weyens et al. 2009). Rhizosphere microorganisms are of major interest due to their

beneficial or detrimental effects on plant growth. It is therefore, important to

understand the mechanism by which rhizosphere microorganisms impact plant

growth in order to develop technologies that could enhance their activities. Micro-

bial populations present in the rhizosphere of legumes have shown substantial

effects on nodulation by Rhizobium spp. and on subsequent growth and yield of

leguminous crops (Kloepper et al. 1989; Glick 1995). Microorganisms inhabiting

rhizosphere of legumes may benefit plants in a variety of ways, like increased

recycling, mineralization and uptake of nutrients; synthesizing vitamins, amino

acids, auxins, gibberlins and plant growth regulating substances; reducing metal

toxicity (bioremediation) in contaminated soils; antagonism with potential plant

pathogens through competition and development of amensal relationships based on

production of antibiotics, siderophores, and/or hydrolytic enzymes (Stockwell and

Stack 2007; Sindhu et al. 2009c).

9.2.1 Increased Recycling, Mineralization and Uptakeof Nutrients

Microorganisms in the rhizosphere influence the availability of mineral nutrients to

the plants, sometimes by increasing the availability of inorganic nutrients to the

plant, and in other cases, using limiting concentrations of inorganic nutrients before

they could reach plant roots. Some rhizosphere bacteria, i.e., rhizobia, azotobacters,

and azospirilla have the ability to fix atmospheric N into plant utilizable form,

ammonia (Franche et al. 2009). Other microorganisms help plants by solubilizing

bound P (Vessey 2003) and potassium or by providing iron and sulfur (Crowley

et al. 1991; Scherer and Lange 1996; Crowley and Kraemer 2007).

9.2.1.1 Biological Nitrogen-Fixing Bacteria and Inoculation Responses

Sustainable agriculture involves the successful management of agricultural

resources to satisfy the changing human needs, while maintaining or enhancing

9 Growth Promotion of Legumes by Inoculation of Rhizosphere Bacteria 197

the environmental quality and conserving natural resources. Consequently,

sustainability considerations demand that alternatives to nitrogen fertilizers are

sought. In this context, BNF offers an alternative in farming practices as it

exploits the capacity of certain N2-fixing bacteria to reduce atmospheric nitrogen

into a compound (ammonia) mediated by enzyme nitrogenase (Bohlool et al.

1992; Burris and Roberts 1993). Legume crops meet their N requirement through

symbiotic nitrogen fixation by forming root nodules with rhizobia (Brewin

2002; Gage 2004) which in turn reduce the dependency of agricultural crops

on fossil fuel-derived nitrogenous fertilizers. Additionally, biologically fixed

N is bound in soil organic matter and thus is much less susceptible to soil

chemical transformations and physical factors that lead to volatilization and

leaching. Therefore, BNF has an important role in sustaining productivity of

soils.

Only some prokaryotes, a few bacteria and cyanobacteria, have acquired the

ability to reduce atmospheric dinitrogen and add this essential nutrient to agricul-

tural soils. Biological N2 fixation occurs in a free-living state, in association with or

in symbiosis with plants. Different N2-fixing bacteria have been used to improve the

supply of fixed N as nutrient to crop plants. Among the nitrogen-fixing systems, the

legume-Rhizobium symbiosis alone accounts for 70–80% of the total N fixed

biologically on global basis per annum and one-third of the total N input needed

for world agriculture. The symbiotic rhizobia have been found to fix N ranging from

57 to 600 kg ha�1 annually (Elkan 1992). Annual inputs of fixed nitrogen are

calculated to be 2.95 million tonnes (Tg) for the pulses and 18.5 Tg for the oilseed

legumes (Herridge et al. 2008).

Rhizobium includes the fast-growing species, Bradyrhizobium includes slow-

growing species and Azorhizobium includes those fast-growing species capable of

forming both stem and root nodules on tropical water-logged legume, Sesbania.Chen et al. (1995) proposed a separate genus, Mesorhizobium, to indicate a growth

rate intermediate between that of Bradyrhizobium strains and typical fast-growing

Rhizobium strains. Subsequently, it was used to denote a phylogenetic position for

rhizobia intermediate between these two genera. According to current taxonomic

classification of root-nodule bacteria, 11 genera and 45 species have been defined

(Sahgal and Johri 2006; Wiliems 2006).

In the rhizosphere of legumes and cereals, other diazotrophic bacteria could also

contribute N to plants. Free-living diazotrophic bacteria contribute upto 15 kg

ha�1 year�1 fixed N and the root-associative bacteria fix N to a level of 15–

36 kg ha�1 year�1. Similarly, cyanobacteria, the free-living nitrogen fixers contri-

bute about one-third of the N requirement of the crop and add about 15–80 kg ha�1

year�1 to the rice cropping system (Elkan 1992) (Table 9.1). The free-living/

associative diazotrophs, although have limited potential in terms of average N

input on acreage basis but inhabit almost all ecological environments and contribute

more in nutrient use efficiency and improvement in crop physiology (Pandey

and Kumar 1989; Wani 1990; Fujiata et al. 1992).

The symbiotic effectiveness of different legume species and their microsym-

bionts has been found to be variable. In general, faba bean (Vicia faba), and


pigeonpea (Cajanus cajan) have been found to be very efficient; soybean (Glycinemax), groundnut (Arachis hypogaea) and cowpea (Vigna sinensis) to be average;

common bean and pea poor in fixing atmospheric N (Hardarson 1993). Among the

legumes, soybean is the dominant crop legume, representing 50% of the global

crop legume area and able to fix 16.4 million tones N annually, representing 77%

of the N fixed by the legumes (Herridge et al. 2008). Inoculation of legumes with

efficient strains of rhizobia has often resulted in significant increases in yields of

various legume crops (Thies et al. 1991; Wani et al. 2007; Franche et al. 2009).

Elsiddig et al. (1999) studied the inoculation effect of Bradyrhizobium strains

TAL 169 and TAL 1371 (introduced) and strains ENRRI 16A and ENRRI 16C

(local) on five guar (Cyamopsis tetragonoloba) cultivars in a field experiment.

Most of the Bradyrhizobium strains significantly increased yield, protein, crude

fiber and mineral content. The locally-isolated strains affected these parameters

more than the introduced ones. Karasu et al. (2009) observed that inoculation

of chickpea (Cicer arietinum) seeds with R. ciceri isolate had a significant effect

on seed yield, plant height, first pod height, number of pods per plant, number

of seeds per plant, harvest index and 1,000 seed weight. But, nitrogen doses

(applied at 0, 30, 60, 90, and 120 kg ha�1 level as ammonium nitrate) had no

significant effect on yield and yield components. Local population genotype as

crop material gave the highest yield (2,149.1 kg ha�1) among three chickpea

genotypes used.

Sindhu et al. (1992) compared the potential of N fixed by Rhizobium strains in

chickpea using non-nodulating genotype PM233 derived from normal nodulating

genotype ICC640. The N fixed by the Rhizobium strains Ca534 and Ca219 in parent

cultivar gave the plant dry weights more than those obtained by applying urea

(80 kg N ha�1) in the non-nodulating mutant PM233, suggesting that in chickpea

effective symbiosis with rhizobia provides more than 80 kg N ha�1. The benefits

of N fixed in legumes to subsequent cereal crops are substantial and persist for

several years due to progressively slow mineralization. The benefits obtained were of

higher magnitude with green manuring crops and upto 532 kg N could be incor-

porated by 60 days green manuring crops where the rate of N accumulation were

rapid upto 10.8 kg N ha�1 day�1 (Peoples and Herridge 1990).

In coinoculation experiments of N2-fixing Azotobacter vinelandii with Rhizo-bium spp., it was found that coinoculation increased the number of nodules on the

Table 9.1 Estimated average rates of biological nitrogen fixation by diazotrophs and associations

Organism/system N2 fixed (kg ha�1 year�1)

Free living microorganisms – Azotobacter, Clostridium and Derxia 0.1–15

Associative symbioses – Azoarcus and Azospirillum 5–25

Cyanobacteria – Nostoc, Anabaena, and Oscillatoria 15–80

Azolla–Anabaena symbiosis 313

Rhizobium–legume symbiosis 57–600

Nodulated non-legumes – Casuarina–Frankia, Parasponia–Rhizobium

2–300


roots of soybean, pea (Pisum sativum) and clover (Trifolium pratense) (Burns et al.1981). Increased nodulation of soybean also occurred in field trial. Similarly,

coinoculation of Azospirillum brasilensewith Rhizobium strains showed synergistic

effect on soybean and groundnut (Iruthayathas et al. 1983; Raverkar and Konde

1988). Compared to single Rhizobium inoculation, coinoculation of Rhizobium spp.

and Azospirillum spp. was found more effective in enhancing the number of root

hairs, the amount of flavonoids exuded by the roots and the number of nodules

(Itzigsohn et al. 1993; Burdman et al. 1997; Remans et al. 2007, 2008b). The effect

of Azospirillum on the legume-Rhizobium symbiosis was found to depend on the

host genotype used. It was observed that Azospirillum–Rhizobium coinoculation

increased the amount of fixed N and the yield of DOR364 genotype of common

bean (Phaseolus vulgaris L.) across all sites on-farm field experiments, whereas a

negative effect of Azospirillum–Rhizobium coinoculation on yield and N2-fixation

was observed in BAT 477 genotype on most of the sites as compared to sole

application of Rhizobium (Remans et al. 2008b).

Field and greenhouse data indicated that increased nodulation of beans

(Phaseolus vulgaris) by R. phaseoli occurred with coinoculation of Pseudomonasputida (Grimes and Mount 1984). However, bean yield and shoot weight were not

significantly affected by coinoculation, demonstrating that increasing nodule num-

ber or infection by Rhizobium spp. may not affect plant productivity. Bolton et al.

(1990) also demonstrated that nodulation of pea increased following the inoculation

of mixtures of R. leguminosarum and a deleterious toxin-releasing Pseudomonassp. However, nodules and dry matter accumulation in shoots were the same whether

or not the Pseudomonas sp. was coinoculated. On the other hand, enhancement in

nodulation, root length, plant biomass and yield by mixed inoculation of rhizobia

with other rhizobacteria in different legumes have been reported. For example,

Chanway et al. (1989) tested nine PGPR strains against single cultivar of lentil and

pea in the field. None of the strains stimulated the growth of pea, but in lentil plots

inoculated with one or more rhizobacterial strains, there were significant increase

in emergence, vigor, nodulation, acetylene reduction activity and root weight. The

enhanced nodulation and growth of chickpea along with reduction in wilt incidence

was observed on coinoculation of rhizobacteria obtained from chickpea rhizosphere

when these strains were coinoculated with an effective R. ciceri strain Ca181 (Khotet al. 1996).

Coinoculation of the five plant growth-promoting fluorescent pseudomonad

strains, isolated from Indian and Swedish soils, and R. leguminosarum bv. viceaestrains, recovered from Swedish soils, improved growth of pea cv. Capella (Dileep

Kumar et al. 2001). In a similar study, Goel et al. (2002) observed that coinocula-

tion of chickpea with Pseudomonas strains MRS23 and CRP55b, and Mesorhi-zobium sp. cicer strain Ca181 increased the formation of nodules by 68.2–115.4%,

at 80 and 100 days after planting as compared to single inoculation of Mesorhizo-bium strain under sterile conditions. The shoot dry weight ratios of coinoculated

treatments at different stages of plant growth varied from 1.18 to 1.35 times that of

Mesorhizobium-inoculated and 3.25–4.06 times those of uninoculated plants.

Similar synergistic effects on nodulation and plant growth have also been observed


for other legumes by dual inoculation of B. japonicum and P. fluorescens in soy-

bean (Li and Alexander 1988; Nishijima et al. 1988; Dashti et al. 1998), R. melilotiwith Pseudomonas in alfalfa (Li and Alexander 1988; Knight and Langston-

Unkeffer 1988), R. leguminosarum with an antibiotic-producing P. fluorescensstrain F113 in pea (Andrade et al. 1998) and Mesorhizobium/Bradyrhizobiumstrains with Pseudomonas sp. in greengram [Vigna radiata (L.) wilczek] and

chickpea (Sindhu et al. 1999; Goel et al. 2000, 2002).

Similarly, bacterization of Bacillus species to seeds or roots altered the compo-

sition of rhizosphere, leading to increase in growth and yield of different legume

crops (Holl et al. 1988). For instance, Halverson and Handelsman (1991) observed

that seed treatment with B. cereus UW85 had 31–133% more nodules than

untreated soybean plants after 28 and 35 days of planting in the field. In the

growth chamber, in sterilized soil-vermiculite mixtures, UW85 seed treatments

enhanced nodulation by 34–61% at 28 days after planting. It was suggested that

UW85 affected the nodulation process soon after planting by stimulating bradyr-

hizobial infections or by suppressing the abortion of infections. In a follow up

study, Turner and Backman (1991) reported that coating of peanut seeds with

B. subtilis improved germination and emergence, enhanced nodulation by Rhizo-bium spp., enhanced plant nutrition, reduced levels of root cankers caused by

Rhizoctonia solani AG-4 and increased root growth. In a similar study, Srinivasan

et al. (1997) reported enhanced nodulation in Phaseolus vulgaris when coinocu-

lated with R. etli strain TAL182 and B. megaterium S49. The mixed inoculation

increased root hair proliferation and lateral root formation. The potential of

Bacillus sp. to enhance nodulation, plant dry matter and grain yield on coinocula-

tion with rhizobia has also been reported for pigeonpea (Podile 1995) and white

clover (Holl et al. 1988). Sindhu et al. (2002a) found that coinoculation of

Bacillus strains with effective Bradyrhizobium strain S24 caused enhancement

in shoot dry mass of green gram ranging from 1.28 to 3.55 at 40 days of plant

growth. Nodule promoting effect and increase in nitrogenase activity was also

observed with majority of Bacillus strains at 40 days of plant growth.

Mishra et al. (2009a) showed that plant growth-promoting bacteria (PGPB)

strain B. thuringiensis-KR1, originally isolated from the nodules of Kudzu vine

(Pueraria thunbergiana), promoted plant growth of field pea and lentil (Lensculinaris L.) when coinoculated with R. leguminosarum-PR1 under Jensen’s tube,

growth pouch and non-sterile soil, respectively. Coinoculation with B. thuringiensis-KR1 (at a cell density of 106 c.f.u. ml�1) had the highest and most consistent

increase in nodule numbers, shoot weight, root weight, and total biomass, over

rhizobial inoculation alone. The enhancement in nodulation due to coinoculation

was 85 and 73% in pea and lentil, respectively, compared to R. leguminosarum-PR1treatment alone. The shoot dry-weight gains on coinoculation with variable cell

populations of B. thuringiensis-KR1 varied from 1.04 to 1.15 times and 1.03–1.06

times in pea and lentil, respectively to those of R. leguminosarum-PR1 inoculated

treatment at 42 days of plant growth. The cell densities higher than 106 c.f.u. ml�1

had an inhibitory effect on nodulation and plant growth whereas lower inoculum

levels resulted in decreased cell recovery and plant growth performance. Similarly,


enhanced nodule number and biomass yield were achieved after coinoculation of

soybean with the B. japonicum SB1 and the plant growth-promoting B. thuringiensis-KR1 (Mishra et al. 2009b).

Inoculation of legumes with different rhizobial strains in general results in a

10–15% increase in yield of legumes. However, the desired impact of biofertilizer

on legumes is usually not achieved under certain field conditions. The inoculation

with commercial inoculants often fails to improve crop productivity (van Elsas and

Heijnen 1990) probably due to the inability of rhizobial species to compete with the

indigenous, ineffective and built in populations, which presents a competitive

barrier to the introduced strains (Sindhu and Dadarwal 2000). In contrast, produc-

tion of bacteriocins by rhizobia have been shown to suppress growth as well as

nodulation by the indigenous non-producer strains, thus improving nodulation

competitiveness of bacteriocin-producing inoculant strains (Goel et al. 1999;

Sindhu and Dadarwal 2000). Transfer and expression of tfx genes (involved in

trifolitoxin production) in various rhizobia showed stable trifolitoxin production

and restricted nodulation by indigenous trifolitoxin-sensitive strains on many

leguminous species (Triplett 1988, 1990). However, attempts to manipulate certain

rhizobial genes in specific legume rhizosphere niches for improving competition

have not been impressive (Nambiar et al. 1990; Sitrit et al. 1993; Krishnan et al.

1999).

Biotechnological approaches used to enhance N2 fixation and crop productivity

(Pau 1991; Hardarson 1993; Sindhu et al. 2009a) under field conditions have been

of limited use. For example, recombinant constructs of R. meliloti and B. japonicumhaving increased expression of nifA and dctA genes although showed increase in

the rate of N fixed but under field conditions, the same constructs did not show any

significant increase in N2-fixation or yields (Ronson et al. 1990). Manipulations of

common nodulation genes to improve the bacterial competition have usually

resulted in either no nodulation, delayed nodulation or inefficient nodulation

(Devine and Kuykendall 1996). Mendoza et al. (1995) enhanced NH4+ assimilat-

ing enzymes in R. etli through genetic engineering, by inserting an additional

copy of glutamate dehydrogenase (GDH), which resulted in total inhibition of

nodulation on bean plants. However, nodule inhibition effect was overcome when

gdhA expression was controlled by NifA and thereby, delaying the onset of GDH

activity after nodule establishment (Mendoza et al. 1998). Similarly, attempts to

engineer hydrogen uptake (Hup+) ability by cloning hydrogenase genes into Hup-

strains of Rhizobium resulted in experimental successes only in areas where

soybeans are cultivated and where the photosynthetic energy is limited (Evans

et al. 1987). Attempts to develop self-fertilizing crops for N have also been a

failure, mainly because of the complexity of the nitrogenase enzyme complex to

be expressed in the absence of an oxygen protection system in eukaryotes (Dixon

et al. 1997). Moreover, induction of nodule-like structures or pseudonodules

using lytic enzymes or hormones treatment in wheat (Triticum aestivum) and

rice (Oryza sativa) though showed nitrogenase activity and 15N2 incorporation,

but the activity expressed was >1% of the value observed for legumes (Cocking

et al. 1994).


9.2.1.2 Phosphate Solubilization and Mobilization and its Agronomic

Significance

In agriculture, phosphorus (P) is second only to N in terms of quantitative require-

ment for crop plants (Goldstein 1986; Fernandez et al. 2007). It is found in soil,

plants and microorganisms in both organic and inorganic forms. However, the total

P content in an average soil is 0.05% and only a very small fraction (�0.1%) of the

total P present in the soil is available to the plants because of its chemical fixation

and low solubility (Stevenson and Cole 1999). The pool of immediately available P

is thus, extremely small and must be supplied regularly to offset plant demands

(Bieleski 1973). Phosphorus may be added to soil either as chemical fertilizers or as

leaf litter, plant residues or animal remains. The P fertilizers are the world’s second

largest bulk chemicals used in agriculture and therefore, the second most widely

applied fertilizer (Goldstein et al. 1993; Goldstein 2007). However, 75% of phos-

phate fertilizers applied to soil are rapidly immobilized and thus become unavail-

able to plants (Rodriguez and Fraga 1999). Therefore, P deficiency is a major

constraint to crop production and under such conditions, the microorganisms

offer a biological rescue system capable of solubilizing the insoluble inorganic

P of soil to make it available to plants and thus maintain the soil health and quality

(Rodriguez and Fraga 1999; Richardson 2001; Deubel and Merbach 2005; Chen

et al. 2006; Khan et al. 2007).

Phosphate solubilizing (PS) and mobilizing microorganisms include bacteria,

actinomycetes as well as the fungi. The most important PS bacteria (PSB) belong to

genera Bacillus and Pseudomonas, though species of Achromobacter, Alcaligenes,Brevibacterium, Corynebacterium, Serratia and Xanthomonas have also been

reported as active P solubilizer (Venkateswarlu et al. 1984; Cattelan et al. 1999;

Khan et al. 2007). In a study, Naik et al. (2008) screened 443 fluorescent pseudo-

monad strains for the solubilization of tricalcium phosphate(TCP) and reported that

18% formed visible dissolution halos on Pikovskaya agar medium plates. Based on

phenotypic characterization and 16S rRNA gene phylogenetic analyses, these strains

were identified as P. aeruginosa, P. mosselii, P. monteilii, P. plecoglosscida,P. putida, P. fulva and P. fluorescens. The P-solubilizing Bacillus species isolatedfrom the rhizosphere of legumes and cereals included B. subtilis, B. circulans,B. coagulans, B. firmus, B. licheniformis, B. megaterium and B. polymyxa (Gaind

and Gaur 1991; Rajarathinam et al. 1995). Other PSB include species of bacteria

like, A. chroococcum, Burkholderia cepacia, Erwinia herbicola, Enterobacteragglomerans, E. aerogenes, Nitrosomonas, Nitrobacter, Serratia marcescens,Synechococcus sp., Rahnella aquatilis, Micrococcus, Thiobacillus ferroxidans andT. thiooxidans (Banik and Dey 1983; Kim et al. 1998; Bagyaraj et al. 2000).

Rhizobium and Bradyrhizobium strains have also been found to solubilize rock

phosphate (RP) or organic P compounds effectively through the production of

organic acids and/or phosphatases (Halder et al. 1991; Abd-Alla 1994). The various

fungi having efficient PS ability belong to genera Aspergillus, Fusarium,Penicillium and Trichoderma (Rashid et al. 2004; Khan et al. 2010). Ahmad et al.

(2008) reported that out of 72 isolates obtained from rhizosphere soil and root


nodules, solubilization of P was commonly detected in Bacillus (80%) followed by

Azotobacter (74%), Pseudomonas (56%) and Mesorhizobium (17%). The principal

mechanism of increasing P availability is the microbial production of organic acids

that may dissolve appetite, releasing soluble forms of P through acidification of

rhizosphere soil. Additionally, the acidification of the rhizosphere environments

through metabolic production of hydrogen ions alters the pH sufficiently to mobilize

soil minerals (Rodriguez et al. 2006).

Phosphatic biofertilizers were first prepared in USSR, using B. megaterium var.

phosphaticum as PSB and the product was named as “phosphobacterin.” It was

extensively used in collective farming for seed and soil inoculation, and reported to

give 5–10% increase in crop yields. Subsequently, inoculation experiments using

phosphobacterin and other PSM for legumes like, groundnut, peas, and soybean

showed an average 10–15% increase in yields in about 30% of the trials (Agasimani

et al. 1994; Dubey 1997; Vessey 2003). The variations under field conditions are

expected due to the effect of various environmental conditions and survival of the

inoculant strains in soil. The inoculation of PSB along with Rock phosphate (RP)

also resulted in increased availability of P for plant utilization (Jisha and Alagawadi

1996). It was observed that inoculation of mineral phosphate solubilizing bacteria

(MPSB) along with 17.5 kg P ha�1 as Massourie rock phosphate (MRP) increased

dry matter in chickpea and was as effective as single super phosphate (Prabhakar

and Saraf 1990). Saraf et al. (1997) showed that PSB inoculation increased seed

yield (10.3 q ha�1) of chickpea as compared to control (8.8 q ha�1). Increased grain

yield (14%) and uptake of N and P was reported in chickpea by inoculation of PSB

along with P fertilizers. The grain and straw yield of chickpea was found to increase

with increasing levels of P (0–60 kg P2O5 ha�1) which further improved by

inoculation of PSB (Sarawgi et al. 1999, 2000). Plant growth-promoting fluorescent

pseudomonad isolate PGPR1, which produced siderophore and indole acetic acid,

and solubilized TCP under in vitro conditions, significantly enhanced the pod yield

(23–26%, respectively), haulm yield and nodule dry weight over the control during

3 years.

Phosphorus deficiency has a negative effect on BNF and the impaired BNF in

P-deficient plants is usually explained by an effect of the low P supply on the

growth of the host plant, on the growth and functioning of the nodule, or on the

growth of both plant and the nodule (Christiansen and Graham 2002). Some

particular strategies have been adopted for the adaptation of nodulated legumes to

limited P supply, such as the maintenance of concentrations of P in nodules much

higher than in other organs (Pereira and Bliss 1987), higher absorption of P from the

solution directly by the nodules and bacteroids (Al-Niemi et al. 1998), increased

N2-fixation per unit of nodule mass to compensate for reduced nodulation,

(Almeida et al. 2000) and higher accumulation of soluble sugars in nodules than

in roots and shoots (Olivera et al. 2004). Araujo et al. (2008) observed an increase in

the activities of acid phosphatases and phytases in nodules of common bean

genotypes at different levels of P supply indicating that this increase in activities

may constitute an adaptive mechanism for N2-fixing legumes to tolerate P defi-

ciency. Similarly, plants grown at limited P supply can increase the activities of


phosphatases and phytases in roots to hydrolyze organic-P compounds in the soil,

thus improving plant P acquisition.

Synergistic effect was observed after coinoculation of N2-fixing bacteria with

PSB. For example, the composite application of P. putida and R. phaseoli increasedP availability to common bean plants and enhanced nodulation of common bean

(Grimes and Mount 1984). The seed inoculation with thermo-tolerant PSB, viz.

B. subtilis, B. circulans and A. niger was found to improve nodulation, available

P2O5 content of soil, root and shoot biomass, straw and grain yield, P and N uptake

by mungbean (Gaind and Gaur 1991). High pod yield and P uptake in groundnut

due to inoculation of P. striata were also recorded (Agasimani et al. 1994).

Increased nodulation, yield attributes, seed index and seed yield of rainfed soybean

were also reported with combined inoculation of P. striata and B. japonicum(Dubey 1997). Similarly, a significant increase in nitrogenase activity, plant growth

and grain yield of pea was found following dual inoculation of R. leguminosarumand PSB (Srivastava et al. 1998).

Attempts to express the mineral phosphate solubilization (MPS) genes in a

different host were found to be influenced by the genetic background of the recipient

strain, the copy number of the plasmids present and metabolic interactions. Thus,

genetic transfer of any isolated gene involved in MPS to improve P-dissolving

capacity in PGPR strains, is an interesting approach. An attempt to improve mps

in PGPR strains, using a PQQ synthase gene from E. herbicola was carried out

(Rodriguez et al. 2000). This gene was subcloned in a broad-host range vector

pKT230. The recombinant plasmid was expressed in E. coli and transferred to PGPRstrains of Burkholderia cepacia and P. aeruginosa, using tri-parental conjugation.

Several of the exconjugants showed a larger clearing halo on medium plates contain-

ing TCP as the sole P source. This experiment indicated the heterologous expression

of this gene in the recombinant strains and improved MPS ability of PGPR.

9.2.1.3 Mineralization of Potassium, Iron and Sulfur Nutrients

in the Rhizosphere

Potassium (K) is the third major essential nutrient for plant growth. It plays an

essential role in enzyme activation, protein synthesis and photosynthesis. Potassium

in soil is present in water-soluble (solution K), exchangeable, non-exchangeable

and structural or mineral forms. Of these, water-soluble and exchangeable pools are

directly available for plant uptake. At low levels of exchangeable K in certain soils,

non-exchangeable K can also contribute significantly to the plant uptake (Memon

et al. 1988). India ranks fourth after USA, China and Brazil in terms of total

consumption of K-fertilizers. Some microorganisms in the soil are able to solubilize

“unavailable” forms of K-bearing minerals, such as micas, illite and orthoclase, by

excreting organic acids which either directly dissolves rock K or chelate silicon

ions to bring the K into solution (Barker et al. 1998; Bennett et al. 1998). These

microorganisms are commonly known as potassium-solubilizing bacteria (KSB) or

potassium-dissolving bacteria or silicate-dissolving bacteria whose application is


termed as “biological potassium biofertilizer (BPF)”. It was shown that KSB

increased K availability in soils and increased mineral uptake by plant (Sheng

and Huang 2002; Sheng et al. 2002, 2003). Therefore, application of KSB holds a

promise for increasing K availability in soils.

In Egypt, some studies were conducted on potassium-dissolving bacteria which

were mainly concentrated on their K releasing capacity along with their effects on

growth and K uptake of the treated plants. In a trial conducted by Balabel-Naglaa

(1997), there were positive responses of broad bean to inoculation with some

species of Bacillus (K releasing bacteria). These positive responses were obvious

on dry weight of shoot and root, nodule number and dry mass of nodules, nitroge-

nase activity, N, P, K contents of foliage, number as well as dry weight of pods,

seed and straw yields. Hu et al. (2006) isolated two phosphate-and potassium-

solubilizing strains, KNP413 and KNP414 from the soil of Tianmu Mountain,

Zhejiang Province (China). Both isolates actively dissolved mineral P and K,

while strain KNP414 showed higher dissolution capacity even than Bacillus muci-laginosus AS1.153, the inoculants of potassium fertilizer widely used in China. In

another study, Lian et al. (2008) studied the mechanism for the release of mineralic

potassium using a thermophilic fungus Aspergillus fumigatus. The thermophilic

fungus A. fumigatus promoted potassium release by means of at least three likely

routes, firstly, by complexing soluble organic ligands, secondly, appealing to the

immobile biopolymers such as the insoluble components of secretion and thirdly,

involving mechanical forces in association with the direct physical contact between

cells and mineral particles.

Iron is yet another essential nutrient and is abundant in soil but most of it is found

in the insoluble form, ferric hydroxide. Thus, iron is only available to organisms at

concentrations at or below 10�18 M in soil solutions at neutral pH. To cope up with

its solubility, many microorganisms synthesize extracellular, low molecular

weight, high affinity Fe3+ chelators commonly referred to as siderophores, in

response to iron stress (Neilands 1981; Neilands and Nakamura 1991) that transport

iron into bacterial cells. Fuhrmann and Wollum (1989) detected a decrease in the

number of taproot nodules and in seedling emergence of soybean and altered

nodulation competition among B. japonicum strains when coinoculated with

Pseudomonas spp. Iron availability was implicated as a factor involved in the

plant-B. japonicum-rhizosphere microflora interactions. Thus, rhizobacteria help

plants in absorbing iron from the soil. The metal-chelating agents produced by

rhizobacteria also play an important role in the acquisition of heavy metals (Leong

1986). These organic substances scavenge Fe3+ and significantly enhance the bio-

availability of soil bound iron (Kanazawa et al. 1994) and regulate the availability of

iron in the plant rhizosphere (Bar-Ness et al. 1992; Loper and Henkels 1999). The

competition for iron in the rhizosphere is controlled by the affinity of the siderophore

for iron, and the probable availability of iron to the microorganisms ultimately

decides the rhizosphere population structure. The concentration of various types of

siderophores, kinetics of exchange and availability of Fe-complexes to microbes as

well as plants has been found to control the binding affinity of siderophore (Loper

and Henkels 1999). Interestingly, the binding affinity of phytosiderophores for iron


is less than the affinity of microbial siderophores, but plants require a lower iron

concentration for normal growth than microbes do (Meyer 2000).

Masalha et al. (2000) reported that plants grown under non-sterile soil systems

were better in terms of iron nutrition than those grown under sterile conditions.

Their data emphasized the role of microbial community on the iron nutrition of

plants. It has been demonstrated that plants grown in metal-contaminated soils are

often iron deficient and the production of siderophores by plant growth-promoting

bacteria may help plants to obtain sufficient iron (Wallace et al. 1992; Burd et al.

2000). In fact, there is evidence that at least part of the toxic effects of some heavy

metals in plants results from an induced iron deficiency and since bacterial

siderophores could provide iron to various plants (Bar-Ness et al. 1991; Wang

et al. 1993), therefore, siderophores produced by rhizobacteria may reduce nickel

toxicity by supplying the plant with iron and hence reduce the severity of nickel

toxicity (Bollard 1983; Bingham et al. 1986).

Another plant nutrient, sulfur (S), is the ninth and least abundant essential

macronutrient. Its uptake and assimilation is crucial because of the key role played

by the S containing aminoacids, methionine and cysteine in maintaining protein

structure and because of its role in plant defense (Rasch and Wachter 2005). Sulfur

atoms are widely distributed in soil and are found in a wide variety of organic and

inorganic forms. These atoms are an integral component of soil humus, plants,

microbial biomass and minerals. Scherer (2009) reported that sulfate (SO42�),

which is a direct source of sulfur for plants, contributed up to 5% of total soil

S; generally more than 95% of soils S are organically bound. Sulfur containing

minerals include pyrite (FeS2) which occurs in ingenious rocks, gypsum

(CaSO4.2H2O) and epsonite (MgSO4.7H2O). Despite its abundance in the earth’s

crust, S is often present in suboptimal quantities in soil or either in unavailable

states. Moreover, the decrease of S input from atmospheric depositions has led to

S deficiency of crops over the past two decades on a worldwide scale that reduced

yield and affected the quality of harvested products. Especially in Western

European countries, incidence of S deficiency has increasingly been reported in

oilseed rape, which is an S demanding plant (Fismes et al. 2000). Therefore, more

attention should be paid to the optimization of S fertilizer application, in order to

cover plant S requirements whilst minimizing environmental impacts.

Sulfur turnover involves both biochemical and biological mineralization

(Gharmakher et al. 2008). Biochemical mineralization, which is the release of

SO42� from the ester sulfate pool through enzymatic hydrolysis, is controlled by

S supply, while the biological mineralization is driven by the microbial need for

organic C to provide energy. The biological oxidation of elemental S and inorganic

S compounds such as H2S, sulphite and thiosulphate is brought about by chemo-

autotrophic bacteria and photosynthetic bacteria. Sulfur-oxidizing bacteria include

Beggiotoa, Chromatium, Chlorobium, Thiobacillus, Sulfolobus, Thiospira and

Thiomicrospira. The species of Bacillus, Pseudomonas, Arthrobacter and Flavo-bacterium are also reported to oxidize elemental S or thiosulphate to sulfate. Under

anaerobic conditions, sulfate is reduced to H2S by sulfate-reducing micro-

organisms, mostly the bacteria. Many bacteria including species of Bacillus and


Pseudomonas are known to reduce S or sulfate to H2S, but among these Desulpho-vibrio desulfuricans and Desulfotomaculum spp. are important.

Nitrogen fixation appears to be affected by S fertilization in faba bean, lucerne,

pea and in red clover (DeBoer andDuke 1982; Scherer and Lange 1996; Habtemichial

and Singh 2007). An important link between S and N nutrition was found in white

clover, lucerne and pea (Zhao et al. 1999; Varin et al. 2009) and sulfur fertilization

was found to stimulate N2 fixation strongly. Scherer et al. (2008) observed that

the amount of leghaemoglobin was reduced by S deficiency in peas and alfalfa,

when no S was added and nodules devoid of leghaemoglobin were more numerous.

Varin et al. (2008) analyzed a set of functional traits in three white clover lines

along a gradient of N and S fertilization on a poor soil. Nitrogen was found to be the

most limiting factor for the VLF (very low fixation) line. S was the element that

modulated the most traits for the nitrogen fixing lines NNU (normal nitrate uptake)

and LNU (low nitrate uptake). Nitrogen fertilization was found to inhibit N2

fixation in clover but N2 fixation was enhanced when S was added. S fertilization

also increased nodule length, as well as the proportion of nodules containing

leghaemoglobin. Thus, sensitivity of white clover to S nutrition would be a disad-

vantage for competition in a situation of sulfur impoverishment.

9.2.2 Synthesis of Auxins, Cytokinins, Gibberlins and Vitamins

Microbial communities of soil and rhizosphere have been found to synthesize auxins,

cytokinins, vitamins and gibberellin-like compounds (Arshad and Frankenberger

1991; Derylo and Skorupska 1993; Patten and Glick 1996; Gutierrez-Manero et al.

2001). These compounds increase the rate of seed germination and stimulate the

development of root tissues leading to an increase in the capacity of the root system

to provide nutrients and water to above ground organs of plants (Arkhipova et al.

2007), and also help the plants to tolerate abiotic stress (Yang et al. 2009). Derylo

and Skorupska (1993) reported that stimulation of clover plant growth under

gnotobiotic conditions resulted from the secretion of water-soluble B vitamins by

fluorescent Pseudomonas sp. strain 267. This was demonstrated by enhancement

of clover growth by naturally auxotrophic strains of R. leguminosarum bv. trifoliiin the presence of the Pseudomonas sp. strain 267 supernatant. The addition of

vitamins to the plant medium increased symbiotic N2-fixation by the clover plants.

Indole acetic acid (IAA) is known as the main auxin in plants and has been

implicated in all aspects of plant growth and development (Taele et al. 2006). The

exposure of plant roots to exogenous microbially produced IAA can affect plant

growth in diverse ways, varying from pathogenesis and growth inhibition to plant

growth stimulation (Spaepen et al. 2007). In fact, low levels of IAA released by

rhizobacteria has been found to promote primary root elongation, whereas, high

levels of IAA stimulated lateral and adventitious root formation (Glick 1995) but

inhibited primary root growth (Xie et al. 1996). Thus, plant growth-promoting

bacteria can facilitate plant growth by altering the hormonal balance within the


affected plant (Lambrecht et al. 2000; Kamnev 2003). Such relationships of rhizo-

bacteria between different crop species could be cultivar or genotype-specific

(Cattelan et al. 1998). For example, the rhizosphere of wheat seedlings harbors a

significant proportion of bacteria that produce phytohormone, indole acetic acid

(IAA), known to increase root growth (Patten and Glick 2002). Moreover, differen-

tial response of inoculation was observed in two genotypes of common bean with

A. brasilense Sp245 mutant strain having reduced auxin biosynthesis or to addition

of increasing concentrations of exogenous auxin (Remans et al. 2008a). Genetic

analysis of recombinant inbred lines revealed two quantitative trait loci (QTLs)

associated with basal root responsive to auxin in common bean.

Although significant and consistent yield increases of rhizobia-inoculated crops

have been attributed to N2 fixation, plant growth regulators may also be involved

(Mayak et al. 1999; Malik and Sindhu 2008). For instance, the rhizobial species are

known to produce IAA in vitro (Bandenoch-Jones et al. 1982; Wang et al. 1982;

Boiero et al. 2007) and nodulated roots often contained substantially greater auxin

concentrations than non-nodulated roots (Dulhart 1967, 1970). Inoculation of

soybeans with spontaneous mutants of Rhizobium japonicum that overproduced

IAA (30-fold more auxin than the wild-type strain) showed a three-fold increase in

the number of root nodules (Kaneshiro and Kwolek 1985). Mutants of B. elkaniistrain deficient in IAA production induced fewer nodules on soybean roots in

comparison to the parental strain and the normal numbers of nodules were reestab-

lished following application of exogenous IAA (Fukuhara et al. 1994). IAA derived

from B. elkanii has been implicated as a causative agent in the swelling of outer

cortical cells of soybean roots and was suggested to provide a competitive advan-

tage for nodulation (Yuhanshi et al. 1995). However, enlargement of cortical cells

was not observed after inoculation with either IAA-deficient mutants of B. elkanii(Yuhanshi et al. 1995) or wild-type B. japonicum strains that do not produce IAA

(Minamisawa and Fukai 1991). Prinsen et al. (1991) demonstrated that flavonoids

released from legume plant roots, which also act as inducers of Rhizobium nodula-

tion genes, stimulated the production of IAA, suggesting that nodule morphogenesis

could be controlled by the highly specific nodulation signal in combination with

phytohormones such as auxins, released by rhizobia.

Coinoculation of legumes with Rhizobium and free-living IAA-producing

bacteria such as Azospirillum brasilense (Yahalom et al. 1990) and several Bacillusspecies (Srinivasan et al. 1996) significantly increased the number of nodules on the

host roots and increased nodule fresh weight and nitrogenase activity, compared

to inoculation with Rhizobium alone. Zhang et al. (1996) reported that Serratiastimulated soybean growth through the production of a plant growth-regulating

compound, which stimulated overall plant vigor and growth, resulting in subse-

quent increase in nitrogen fixation. Mayak et al. (1999) showed that an IAA over

producing mutant of P. putida caused extensive development of adventitious roots

on mung bean cuttings. It was suggested that inoculation with these free-living

bacteria increase the number of infection sites on roots for attachment and nodula-

tion by Rhizobium. In addition, enhanced production of flavonoid-like compounds

or phytoalexins in roots of several crop plants by inoculation of Pseudomonas sp.


(Parmar and Dadarwal 1999; Goel et al. 2001) could induce the transcription of

nodulation (nod) genes (Peter and Verma 1990), leading to increase in nodulation.

In contrast, similar experiments using mutants of B. megaterium with altered IAA

production levels had a negative effect on symbiotic parameters (Srinivasan et al.

1996). Mutants of Pseudomonas strains altered in IAA production were derived by

Tn5 mutagenesis (Malik 2002; Malik and Sindhu 2008). Coinoculation studies of

wild-type Pseudomonas strains with Bradyrhizobium strain S24 and IAA over-

producer Pseudomonas mutants resulted in more nodules in green gram compared

to wild type Bradyrhizobium strain at 50 days of growth. Camerini et al. (2008)

introduced iaaM gene (involved in IAA biosynthesis) from Pseudomonas savastanoiand the tms2 gene from A. tumefaciens into R. leguminosarum bv. viciae LPR1105.Free-living bacteria harboring the promoter iaaM-tms2 construct (strain RD20)

released 14-fold more IAA in the growth medium than the wild-type parental strain

and elicited the development of vetch root nodules containing up to 60-fold more

IAA than nodules infected by the wild-type strain LPR1105. The root nodules elicited

in vetch by RD20, were heavier and had an enlarged and more active meristem, and

showed a significant increase in acetylene reduction activity (ARA).

9.2.3 Effect of Rhizobacteria on Phytoremediation in MetalStressed Soil

Pollution of biosphere by toxic metals has accelerated dramatically since the

beginning of the industrial revolution (Kabata-Pendias and Pendias 1989). Heavy

metal pollution of soil is a significant environmental problem and has its negative

impact on human health and agriculture. The most common heavy metal contami-

nants are Cd, Cr, Cu, Hg, Pb, and Ni. Heavy metals ions, when present at an

elevated level in the environment, are excessively absorbed by roots and translo-

cated to shoot, leading to impaired metabolism and reduced growth (Bingham et al.

1986). In addition, excessive metal concentrations in contaminated soils resulted in

decreased soil microbial activity and soil fertility, and yield losses (McGrath et al.

1995). Phytoremediation has been reported to be an effective, in situ, non-intrusive,

low-cost, ecofriendly, socially accepted technology to remediate polluted soils

(Garbisu et al. 2002). Another alternative is to provide them with an associated

plant growth-promoting rhizobacteria, which also is considered an important com-

ponent of phytoremediation technology (Glick 2003; Jing et al. 2007). Therefore,

the use of rhizobacteria to enhance phytoremediation of soil heavy metals pollution

has recently received more attention (Weyens et al. 2009).

The functioning of associative plant-bacterial symbioses in heavy-metal-

polluted soil can be affected from the side of both the micropartner (plant-

associated bacteria) and the host plant (Glick 1995). Chaudri et al. (1992) found

that Rhizobium populations were reduced at concentrations >7 mg kg�1 soil in

their Cd treatments. Field studies of metal contaminated soils have similarly


demonstrated that elevated metal loadings can result in decreased microbial commu-

nity size (Chander and Brookes 1991). Some rhizobacteria can exude a class of

rhizobacterial secretion, such as IAA, siderophores, 1-aminocyclopropane-1-carbox-

ylic acid (ACC) deaminase which increased bioavailability and facilitated root

absorption of heavy metals, such as Fe (Crowley et al. 1991), enhanced tolerance

of host plants by improving the P absorption (Liu et al. 2000) and promoted plant

growth (Burd et al. 2000; Ellis et al. 2000). Rajkumar et al. (2005) isolated

Pseudomonas sp. strain RNP4 from tannery waste contaminated soil which toler-

ated concentrations up to 450 mg Cr6+ L�1 on a Luria-Bertani (LB) agar medium

and reduced a substantial amount of Cr6+ to Cr3+ in LB liquid medium. The strain

also produced substantial amount of IAA, exhibited the production of siderophores

and solubilized phosphorus. The strain was found to promote the growth of black

gram, Indian mustard and pearl millet in the presence of Cr6+, suggesting the

innate capability of the Pseudomonas isolate for parallel bioremediation and

plant growth promotion. In another study, Safronova et al. (2006) found that

pea plants inoculated with root-associated bacteria containing ACC deaminase

activity produced longer roots, greater root density and improved nutrient uptake

by pea genotypes cultivated in cadmium supplemented soil. Inoculation of pea

plants with a poplar endophyte that degraded 2,4-dichlorophenoxyacetic acid

(2,4-D) resulted in increased removal of 2,4-D from the soil (Germaine et al.

2006). Moreover, the plants did not show toxic responses and did not accumulate

2,4-D in their tissues.

Liu et al. (2007) demonstrated that inoculation of alfalfa with Comamonas sp.strain CNB-1 not only removed 4-chloronitrobenzene (4-CNB) completely within 1

or 2 days from soil but also eliminated the phytotoxicity of 4-CNB to alfalfa plants.

Tank and Saraf (2009) selected five plant growth-promoting bacterial strains based

on their P solubilization ability, IAA production and biocontrol potentials. These

isolates were also able to grow and produced siderophores in presence of heavy

metals like Ni, Zn, and Cd. A positive response of bacterial inoculants was observed

in chickpea plants towards toxic effect of nickel present in soil at different con-

centrations (0, 1 and 2 mM) and bacterial inoculants enhanced fresh and dry weight

of chickpea plants even at 2 mM nickel concentration. The accumulation of nickel

plant�1 was just 50% in Pseudomonas-inoculated plants as compared to uni-

noculated plants with 2 mM nickel concentration along with increased biomass.

The development of engineered endophytic bacteria that improved the phytoreme-

diation of volatile organic compound trichloroethylene (TCE) was found to protect

host plants against the phytotoxicity of TCE and contributed to a significant

decrease in TCE evapotranspiration (Barac et al. 2004). Similarly, the genetic

modification of the polychlorinated biphenyls (PCB)-degrading bacteria Pseudo-monas fluorescens F113, to improve its performance in the rhizosphere, could

be manipulated by improving symbiotic microorganisms. Thus, the rhizoremedia-

tion of PCBs by P. fluorescens was improved in which biphenyl degradation

is regulated using a system that responds to signal from alfalfa roots (Villacieros

et al. 2005).


9.2.4 Rhizobacteria as Biocontrol Agents

The suppression of growth of soil-borne plant pathogens by the use of microorga-

nisms, natural or modified, genes or gene products to reduce the effects of undesir-

able organisms (pests) is referred to as biocontrol. Rhizobacteria inhibit the growth

of various pathogenic bacteria and fungi resulting in suppression of the diseases

caused by such pathogens (Weller 1988; Thomashow and Weller 1996). Disease

suppression by biocontrol agents involves a sustained manifestation of interactions

among the plant, the pathogen, the biocontrol agent, the microbial community on

and around the plant, and the physical environment (Pierson and Weller 1994).

Strains of Pseudomonas fluorescens, P. putida, P. aureofaciens, P. cepacia and

P. aeruginosa have been found to antagonize the growth of pathogens leading to

substantial disease control (Chandra 1997; Weller 2007). Different strains of

Bacillus thruingiensis, B. sphaericus, B. cereus and B. subtilis are also used as

biocontrol agents (Asaka and Shoda 1996; Hervas et al. 1997).

Trapero-Casas et al. (1990) reported that coating of chickpea seeds with the

P. fluorescens (strain Q29z-80) increased the yield which was comparable to those

obtained with any of the fungicide seed treatments used to control seed rot and

preemergence damping-off disease caused by P. ultimum in the field. Hervas et al.

(1997) observed that treatment of B. subtilis, nonpathogenic F. oxysporum and/or

T. harzianum, when applied alone or in combination, to chickpea cultivars “ICCV

4” and “PV 61” could effectively suppress the disease caused by the highly virulent

F. oxysporum f. sp. ciceris. In comparison with the control, the final disease

incidence was reduced by B. subtilis (18–25%) or nonpathogenic F. oxysporum(18%). The extent of disease suppression was higher and more consistent in cultivar

“PV 61” than in “ICCV 4” whether colonized by B. subtilis, non pathogenic

F. oxysporum or T. harzianum. Nautiyal (1997) found that among 478 bacteria

obtained from roots of chickpea rhizosphere by random selection, 44 rifampicin

resistant strains showed biocontrol activity against F. oxysporum f. sp. ciceri,R. bataticola and Pythium sp. under in vitro studies. In a greenhouse test, seed

bacterization of chickpea with P. fluorescens NBRI 1303 increased the germination

of seedlings by 25% reduced the number of diseased plants by 45% as compared

with non-bacterized controls. Significant growth increases in terms of shoot length,

dry weight, and grain yield, averaging 11.6, 17.6, and 22.61%, respectively, above

untreated controls were obtained in field trials.

Plant growth-promoting fluorescent pseudomonad isolate PGPR1, which

produced siderophore and IAA, and solubilized TCP under in vitro conditions,

also suppressed the soil-borne fungal diseases like collar rot of peanut (caused by

A. niger) in field trials (Dey et al. 2004). Jamali et al. (2004) studied effect of seven

antagonistic bacteria on control of Fusarium wilt under green house conditions.

Isolates B-120, B-32, B-28 and B-22 were identified as B. subtilis and isolates

Pf-100, Pf-10 and CHAO were identified as P. fluorescens. Results revealed that

only isolate B-120 reduced Fusarium wilt of chickpea in both seed and soil

treatments. Soil treatment of bacteria showed better effects on plant growth than


that of bacterial seed treatment. Statistically significant biocontrol effects were

observed when lettuce seedlings were inoculated into naturally Rhizoctoniasolani-infested lettuce fields with bacterial suspensions of two endophytic strains,

Serratia plymuthica 3Re4-18 and P. trivialis 3Re2-7 with rhizobacterium P. fluor-escens L13-6-12, 7 days before and five days after planting in the field (Scherwinskiet al. 2008). Usually, no general relationship was observed between the ability of a

bacterium to inhibit a pathogen under in vitro and in situ disease suppression

(Schroth and Hancock 1982; Wong and Baker 1984). Bacterial strains producing

the largest zones of inhibition on agar media do not always make the best biocontrol

agents. Therefore, some in vitro conditions have been modified to more closely

simulate natural conditions (Randhawa and Schaad 1985). Among the numerous

examples of biocontrol agents reported for disease control of soil-borne pathogens,

only few studies provide mechanistic information for the activities of these agents.

Recently, the use of mutants that lack certain in vitro and in situ activities have

provided strong evidence for the involvement of specific molecules in biocontrol.

9.2.4.1 Mechanisms Involved in Biocontrol

For effective biocontrol of plant disease, the rhizobacteria must establish and grow

in an ecological habitat that includes indigenous pathogenic microorganisms. Thus,

root colonization by rhizobacteria appears to be an important factor in biological

control and plant growth promotion. In recent years, tremendous progress has been

made in characterizing the process of root colonization by biocontrol agents, the

biotic and abiotic factors affecting colonization, bacterial traits and genes con-

tributing to pathogen suppression (Benizri et al. 2001; Sindhu et al. 2009b).

Rhizobacteria inhibit the growth of phytopathogenic microorganisms by various

mechanisms.

Competition for Nutrients and Infection Sites

The rhizosphere microflora directly or indirectly inhibit the invasion of pathogen

on plant tissue. Root-inhabiting microorganisms and plant pathogens could com-

pete for space, nutrients or even for binding sites on the root surface. Space and

nutrients competition could result in failure of the pathogen to develop critical

population densities for disease initiation, whereas, the competition for specific

binding sites would reduce the capability of plant pathogen to initiate the infection

process. Pseudomonads possess the capacity to catabolize diverse nutrients and

have fast generation time in the root zone, and hence, they are logical candidates for

competition for nutrients against the slow growing pathogenic fungi and could

result in biological control of pathogens (Weller 1985). Elad and Chet (1987)

carried out a study to evaluate the antagonistic mechanism of rhizobacteria against

damping off disease caused by Pythium. The competition for nutrients between


germinating oospores of Pythium aphanidermatum and biocontrol rhizobacteria

was unique and was correlated significantly with disease suppression.

Interference in Chemotactic Attraction

Crop rotations and tillage management have been shown to influence specific

microbial populations (Sturz et al. 1997). Rhizobacteria could spur a root exudation

response in plants that is species specific (Merharg and Killham 1995). The close

interactions between plants and rhizobacteria encourage the establishment of

specific and beneficial rhizosphere, and such associations between different crop

species could be cultivar-specific. Thus, certain cultivars of clover can foster the

development of rhizo- and endophytic bacteria that favor the growth and develop-

ment of specific cultivars of potatoes (Sturz and Christie 1998). An additional role

of rhizosphere microbes in reducing root disease incidence is in interfering with

chemotactic attraction of the pathogen to root receptor sites. Scher et al. (1985)

suggested that chemotaxis might be the first step in root colonization. A variety of

compounds as components of root exudates may serve as attractants for plant

pathogens. Growth of root inhabitants (including mycorrhizal fungi) necessarily

reduced both the quantity and diversity of organic compounds diffusing from the

root, thereby, diminishing the probability of encounter by a plant pathogen (Davis

et al. 1979).

Antibiotic Production

Antibiotic production by rhizobacteria is one of the major mechanisms postulated

for antifungal activity to suppress pathogens in the rhizosphere and to promote plant

growth. The role of antibiotics in disease suppression has been demonstrated

in many biocontrol systems by mutant analyses and biochemical studies using

purified antibiotics (Stockwell and Stack 2007). These antimicrobial compounds

may act on plant pathogenic fungi by inducing fungistasis, inhibition of spore

germination, lysis of fungal mycelia or by exerting fungicidal effects. A large

number of antibiotics including phenazine carboxylic acid, diacetyl phloroglucinol,

oomycin A, pyocyanine, pyrroles, pyoluteorin, pyrrolnitrin, iturin A, surfactin, etc.

are produced by rhizobacteria (Bender et al. 1999; Sindhu et al. 2009b), which help

in the suppression of pathogen growth. The first antibiotic clearly implicated in

biocontrol by fluorescent pseudomonads was the phenazine derivative that con-

tributed to disease suppression by Pseudomonas fluorescens strain 2-79 and

P. chlororaphis strain 30–84 (formerly P. aureofaciens), which were suppressive

to the take-all disease of wheat roots caused by Gaeumannomyces graminis var.tritici (Gurusiddaiah et al. 1986). The antibiotic was found active against several

fungi including G. graminis var. tritici, R. solani and P. aristesporum. Pseudomonasfluorescens strain CHAO was found to produce a variety of secondary metabolites,

i.e., 2,4-diacetyl phloroglucinol, pyoluteorin, hydrogen cyanide, salicylic acid,


pyochelin and pyoverdine, and protected various plants from diseases caused by

soil borne pathogenic fungi (Stutz et al. 1986). Anjaiah et al. (2003) found that an

isolate of P. aeruginosa PNA1, obtained from chickpea rhizosphere, protected the

plants from Fusarium wilt until maturity in moderately tolerant genotypes of

pigeonpea and chickpea. Root colonization of pigeonpea and chickpea, which

was measured using a lacZ-marked strain of PNA1, showed ten-fold lower root

colonization of susceptible genotypes than that of moderately tolerant genotypes,

indicating that this plant-bacteria interaction could be important for disease sup-

pression in this plant. Its Tn5 mutants (FM29 and FM13), which were deficient in

phenazine production, caused a reduction or loss of wilt disease suppression

in vivo. Similarly, B. cereus strain UW85 suppressed the diseases caused by the

oomycetes. Analysis of B. cereus mutants showed a significant quantitative rela-

tionship between disease suppressiveness and the production of two antibiotics,

zwittermicin A and kanosamine (Silo-Suh et al. 1994; Milner et al. 1996). The

purified antibiotics suppressed the disease and inhibited the development of oomy-

cetes by stunting and deforming germ tubes of germinating cysts. Bacillus subtilisRB14, which produced antibiotics iturin A and surfactin, was found to suppress

damping off disease caused by Rhizoctonia solani (Asaka and Shoda 1996).

Production of Siderophores

Iron is an essential element for all living organisms and most of it is found in the

insoluble form at neutral pH. To cope up with low solubility of iron, many

microorganisms synthesize extracellular Fe3+ chelators i.e., siderophores, in

response to low iron stress (Neilands and Nakamura 1991) that transport iron into

bacterial cells. Plant growth promoting rhizobacteria (PGPR) produced different

types of siderophores, which were involved in disease suppression and plant growth

promotion (Leong 1986). The various categories of siderophores produced by

PGPR include catechol, hydroxamate, pyoverdine, pyochelin, cepabactin, schizo-

kinen and some other types like azotochelin, rhizobactin, anthranilic acid and

azotobactin.

Kloepper et al. (1980) were the first to demonstrate the importance of side-

rophores production in biocontrol of plant pathogens with pseudobactin, a

siderophore produced by Pseudomonas strain B10. The addition of 1.0 mM ferric

chloride to an iron-deficient medium abolished the antagonism under in vitro

conditions and the fluorescence by the PGPR were not observed. Studies with

various siderophore-negative Tn5 mutants showed that pseudobactin of either

pyoverdine and pyochelin type was necessary to achieve wild-type levels of

protection against Pythium-induced damping off disease (Buysens et al. 1996).

Goel et al. (2000) isolated pigment overproducer mutant MRS16M-1 from

Pseudomonas strain MRS16, that was more inhibitory to the fungal pathogens,

whereas, non-producer mutant MRS16M-5 was less inhibitory on nutrient agar

medium. Addition of 100 mM ferric chloride to the medium decreased inhibition of

fungal growth, suggesting the involvement of siderophores and other antifungal


secondary metabolites. Dileep Kumar et al. (2001) found that both the fluorescent

pseudomonads and Rhizobium strains exhibited a wide range of antifungal activ-

ity against pathogens specific to pea. In a synthetic culture medium, all the plant

growth promoting fluorescent pseudomonad strains produced siderophores, which

expressed antifungal and antibacterial activity. Seed bacterization with plant

growth-promoting strains, alone and together with a Rhizobium leguminosarumbiovar viceae isolate, 361–27 reduced the number of infected peas grown in

Fusarium oxysporum infested soils. Seed bacterization with siderophore-producing

P. fluorescens isolates, viz. PGPR1, PGPR2 and PGPR4, suppressed the soil-borne

fungal diseases like collar rot of peanut caused by A. niger and isolate PGPR4

also suppressed stem rot caused by S. rolfsii (Dey et al. 2004).

Production of Hydrolytic Enzymes

Some cell wall lysing enzymes produced by rhizobacteria have been found to

cause the destruction of pathogens. For example, Chet et al. (1990) cloned the

gene encoding chitinase enzyme from S. marcescens and transferred it into

E. coli. The partially purified chitinase caused extensive bursting of the hyphal

tips. This chitinase preparation was effective in reducing disease incidence caused

by R. solani under greenhouse conditions. In other study, Chet et al. (1993)

isolated three different chitinase genes from Serratia, Aeromonas, and Tricho-derma. The cloned genes were expressed in E. coli and subsequently introduced

into R. meliloti, P. putida, and Trichoderma strains resulting in increased

chitinolytic activity of transformants against Sclerotium rolfsii and R. solani.Recombinant strains of R. meliloti were constructed which carried chiA genes

to produce chitinase. The recombinant strain expressed chitinase during symbio-

sis in alfalfa roots (Sitrit et al. 1993). Khot et al. (1996) reported that certain

isolates of Pseudomonas and Bacillus produced chitinase, b-1,3 glucanase (lami-

narinase) and siderophores. Seed inoculation of these bacteria or application of

cell free extract on seed resulted in 48.6 and 31.6% reduction of the wilt incidence

of chickpea under field conditions in a wilt sick nursery. Pseudomonas strains

isolated from the rhizosphere of chickpea and green gram were also found to

produce chitinases and cellulases in culture-free supernatants and inhibited

growth of P. aphanidermatum and R. solani on potato dextrose agar medium

plates (Sindhu and Dadarwal 2001).

Production of Secondary Metabolites

Among other metabolites, hydrogen cyanide (HCN) is produced by many rhizo-

sphere bacteria and has been demonstrated to play a role in biological control of

pathogens (Voisard et al. 1989). HCN over-producing bacterial strains resulted in

small but statistically significant increase in the suppression of symptoms caused by

Mycophaerella graminicola and Puccinia recondita f. sp. tritici on wheat seedling


leaves. Pseudomonas aeruginosa strain zag2 was reported to produce pyocyanin,

siderophore and hydrogen cyanide (Hassanein et al. 2009). The minimum inhibi-

tory concentration of the extracted pigmented compound against Candida albicanswas 40.69 mg ml�1 and the antifungal activity of the compound was remarkable at

100�C for 20 min. The toxic volatile compound HCN produced by the bacteria was

found to reduce the growth of both F. oxysporum and Helminthosporium sp.

whereas A. niger was not affected.The fungal pathogens also cause the plant to synthesize stress ethylene (van

Loon et al. 2006) and much of the damage sustained by plants infected by phyto-

pathogens occurs as a result of the response of the plant to the increased levels of

ethylene (van Loon 1984). It is well known that exogenous ethylene often increases

the severity of fungal infection, while some ethylene synthesis inhibitors signifi-

cantly decrease the severity of a fungal infection (Elad 1990; Robinson et al. 2001).

A number of PGPR, which stimulated root growth of different plant species were

found to contain the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deami-

nase, which hydrolysed the ethylene precursor ACC to ammonia and a-ketobuty-rate, and as a result, decreased ethylene biosynthesis by plants (Glick et al. 1994;

Hall et al. 1996; Belimov et al. 2001). The ACC deaminase-containing biocontrol

bacterial strains were also found more effective than biocontrol strains that did not

possess this enzyme (Wang et al. 2000).

Induction of Systemic Resistance

Some biocontrol agents induced a sustained change in the plant and increased its

tolerance to infection against fungal and bacterial pathogens (Maurhofer et al.

1998), a phenomenon known as induced systemic resistance (ISR). Various non-

pathogenic rhizobacteria have the ability to induce a state of systemic resistance in

plants, which provided protection against a broad spectrum of phytopathogenic

organisms including fungi, bacteria and viruses (Bakker et al. 2007). Induced

resistance brought about by prior inoculation of the host by a pathogen, avirulent

or incompatible forms of a pathogen, or heat killed pathogens has been attributed to

induce physiological response of the host plant against subsequent inoculation by

the virulent pathogens (Hoffland et al. 1996). Induced systemic resistance in plants

has been demonstrated in over 25 crops, including legumes, cereal crops, cucurbits,

solanaceous plants and trees against a wide spectrum of pathogens. The mechanism

of ISR has also been studied in plant growth-promoting Bacillus spp. (Kloepper et al.2004). Bacterial production of the volatile 2,3-butanediol triggered the expression

of Bacillus-mediated ISR in Arabidopsis. The signaling pathway that is activated inthis case depended on ethylene but was independent of salicylic acid and jasmonic

acid signaling (Ryu et al. 2004). Whether or not biocontrol agents suppress disease

by inducing resistance, it is essential that ISR and biocontrol strategies be com-

patible because future agricultural practices are likely to require the integration of

multiple pest control strategies.


9.2.5 Selection of Rhizobacteria with Multiple Plant Growth-Promoting Traits

The use of beneficial soil microorganisms as agricultural inputs for improved crop

production requires selection of rhizosphere-competent microorganisms with plant

growth-promoting attributes. The selection of PGPR strains depends largely on

their growth promoting activities, such as, production of IAA and siderophores,

P solubilization and inhibition of pathogenic microorganisms. However, the pres-

ence of one or all of these traits does not qualify them to be a PGPR for one

particular crop or spectrum of crops. For example, Cattelan et al. (1999) reported

one rhizobacterial isolate which did not share any of the PGPR traits tested in vitro

except antagonism to Sclerotium rolfsii and Sclerotinia sclerotium, but it promoted

soybean growth. This indicates that besides such growth promoting traits, there are

unexplained mechanisms, which also influence the growth of plants and requires

the close proximity of PGPR to the roots of plants. Hence, there is no clear

separation of growth promotion in plants and biological control induced by bacte-

rial inoculants (Lugtenberg et al. 1991; Bloemberg and Lugtenberg 2001). Bacterial

strains selected initially for in vitro antibiosis as part of evaluating biological

control activity frequently demonstrated growth promotion in the absence of target

pathogen (Sindhu et al. 1999; Goel et al. 2002). Similarly, PGPR selected initially

for growth promotion in the absence of pathogens, may demonstrate biological

control activity when challenged with the pathogens, presumably by controlling

deleterious microorganisms or non-target pathogens.

Direct growth promotion occurs when a rhizobacterium produces metabolites

that directly promote plant growth without interactions with native microflora

(Kloepper et al. 1991). Dileep Kumar and Dube (1992) reported that fluorescent

siderophore-producing Pseudomonas strain RBT13, originally isolated from the

tomato roots, enhanced seed germination of chickpea and soybean, and resulted in

increased root and shoot weight as well as yield of the crops. Sindhu et al. (2002b)

reported plant growth promoting effects of fluorescent Pseudomonas sp. on coin-

oculation with Mesorhizobium sp. Cicer strain under sterile and “wilt sick” soil

conditions in chickpea. The coinoculation resulted in enhanced nodulation by

Mesorhizobium sp. and increased shoot dry weight by 3.92–4.20 times in compari-

son to uninoculated controls. Under indirect growth promotion mechanism, the

production of antibiotics, siderophores and HCN by microorganisms decreased the

population and activities of pathogens or deleterious microorganisms and thereby,

increased the plant growth (Pierson and Weller 1994).

Nine different isolates of PGPR (Pseudomonas sp.) were selected from a pool of

233 rhizobacterial isolates obtained from the peanut rhizosphere based on ACC-

deaminase activity (Dey et al. 2004). Four of these isolates, viz. PGPR1, PGPR2,PGPR4 and PGPR7 produced siderophore and IAA. In addition, P. fluorescensPGPR1 also possessed the properties like, P-solubilization, ammonification and

inhibited A. niger and A. flavus under in vitro conditions. In addition to the traits

exhibited by PGPR1, the strain PGPR4 showed strong in vitro inhibition to


S. rolfsii. In field trials on peanut, plant growth-promoting fluorescent pseudomo-

nad isolates, viz. PGPR1, PGPR2 and PGPR4, significantly enhanced the pod yield

(23–26, 24–28, and 18–24%, respectively), haulm yield and nodule dry weight over

the control in 3 years. Inoculation with plant growth-promoting P. fluorescensisolates, viz. PGPR1, PGPR2 and PGPR4, was found to suppress the soil-borne

fungal diseases like collar rot of peanut caused by A. niger and isolate PGPR4 also

suppressed stem rot caused by S. rolfsii. Hynes et al. (2008) screened 563 bacteria

originating from the roots of pea, lentil and chickpea for the suppression of legume

fungal pathogens and for plant growth promotion. Screening of bacteria showed

that 76% isolates produced siderophore, 5% isolates showed ACC deaminase

activity and 7% isolates were capable of indole production. Twenty-six isolates

(5%) suppressed the growth of Pythium species strain p88–p3, 7% suppressed the

growth of Fusarium avenaceum and 9% suppressed the growth of R. solaniCKP7. Four isolates promoted the growth of lentil and one isolate promoted the

growth of pea. Fatty acid profile analysis and 16S rRNA sequencing of the isolates

showed that 39–42% were the members of Pseudomonadaceae and 36–42% of the

Enterobacteriaceae families.

In search of efficient PGPR strains, 72 bacterial isolates were obtained from

different rhizospheric soil and plant root nodules (Ahmad et al. 2008). Of these,

more than 80% of the isolates belonging to genera Azotobacter, Pseudomonas,and Mesorhizobium produced IAA, whereas, only 20% of the Bacillus was IAA

producer. Solubilization of P was commonly detected in the isolates of Bacillus(80%) followed by Azotobacter (74%), Pseudomonas (56%) and Mesorhizobium(17%). All tested isolates produced ammonia. Siderophore production and antifun-

gal activity of these isolates except Mesorhizobium were exhibited by 10–13%

isolates. HCN production was more common trait of Pseudomonas (89%) and

Bacillus (50%). Pseudomonas Ps5 and Bacillus B1 isolates showed broad-spectrumantifungal activity against Aspergillus, Fusarium and Rhizoctonia bataticola.

9.3 Biotic and Abiotic Factors Affecting Rhizosphere

Colonization

It is well established that root colonization by biocontrol agent and beneficial micro-

organisms is a prerequisite to suppress the plant disease and to enhance plant

growth. Root colonization by introduced bacteria could be improved by increasing

the population size, distribution or survival of bacteria, along with manipulation of

soil factors that may positively or negatively affect colonization. Bacterial traits

such as growth rate, cell surface properties, motility (Boelens et al. 1994), chemo-

taxis to root exudates, production of secondary metabolites and tolerance to stressed

environment (e.g., dehydration and temperature) also contributes to rhizosphere

competence. Plant characteristics, like root structure, age and plant genotype as

well as physico-chemical properties of soil, application of pesticides etc. were


found to affect rhizosphere colonization by the beneficial rhizobacteria. Use of green

fluorescent protein (gfp) and in situ monitoring based on confocal laser scanning

microscope (CLSM) could be used to understand the rhizosphere competence and

root colonization (Johri et al. 2003). Using this technique, it was found that the

Pseudomonas (biocontrol strains) colonized the seed and root surface at the same

position, as did the pathogenic fungi that they controlled (Bloemberg et al. 2000).

Another promising option considered important for understanding colonization is to

screen mutants directly. Mutants of Pseudomonas strains of both phenotypes have

been identified and analysis of these mutants indicated that prototrophy for amino

acids and vitamins, rapid growth rate, utilization of organic acids and lipopolysac-

charide properties contributed to colonization ability. Modification of genes involved

in the biocontrol activity of biological control agents also played a key role

in improving the potential rhizosphere competence as well as antifungal activity of

biological control agents (Carroll et al. 1995). Moreover, biocontrol activity of

P. fluorescens carrying PCA coding mini-Tn5 vector was enhanced by introducing

phzH gene from Pseudomonas chlororaphis PCL1391 (Timmis-Wilson et al. 2000).

9.4 Development of Bacterial Inoculants and Constraints

in Their Use

Rhizobium and Bradyrhizobium inoculants have been marketed with success for

over a century. Releases of these nodule-forming microorganisms into soils have

been successful. Inoculation with such inoculants has resulted in their establishment

into soil and onto plant roots to a level sufficiently higher for the intended purpose.

However, the desired impact of biofertilizer application under field conditions has

been variable and inoculation of legume plants with commercial inoculant strains

often fails to improve crop productivity (van Elsas and Heijnen 1990; Akkermans

1994). The problem is of the survival of inoculant diazotrophic bacteria under field

conditions. For each introduction, abiotic soil factors such as texture, pH, tempera-

ture, moisture content and substrate availability need critical assessment since

these factors largely determine the survival and activity of the introduced micro-

organisms (van Veen et al. 1997). In addition, the response of the inoculant to the

prevailing soil conditions also depends on its genetic and physiological constitution

(Brockwell et al. 1995). The use of genetic markers like resistance to antibiotics or

introduction of metabolic markers from other bacterial species could help in tracing

the introduced strains, whether it is rhizobia, cyanobacteria, azotobacters or

azospirilla (Wilson et al. 1995). Another important reason for the inconsistency

observed due to inoculation of PGPR could be the coating of seeds by low number

of rhizobial cells. Higher or lower dosage of PGPR may have a detrimental effect

on nodulation and growth of plant as demonstrated by Plazinski and Rolfe (1985).

On the commercial front, approximately 20 bacterial biocontrol products based

on Pseudomonas, Bacillus, Streptomyces and Agrobacterium strains have been


marketed. The discovery of many traits and genes involved in the beneficial effects

of PGPRs has resulted in a better understanding of the performance of bioinoculants

in the field.

Some of these strains may provide effective control of diseases in certain soils,

in certain geographic regions or on particular crops. Generally, microorganisms

isolated from the rhizosphere of a specific crop are better adapted to that crop and

may provide better control of disease than organisms originally isolated from other

species (Cook 1993). Despite the extensive research where biological agents have

been used to control plant diseases, there have been limited commercial success.

Many biological agents do not perform better in the field due to the complexity and

variability of physical, chemical, microbiological and environmental factors in the

field. Therefore, applications of a mixture of biocontrol agents may be a more

ecologically sound approach because it may result in better colonization and

enhance the level and consistency of disease control by providing multiple mecha-

nisms of action, a more stable rhizosphere community and effectiveness over a

wide range of environmental conditions occurring throughout the growing season.

In addition, the genetic diversity of these strains may be tapped by combining them

in mixed inoculants. Certain mixtures of fluorescent pseudomonads suppressed

disease more effectively than did single-strain inoculants (Pierson and Weller

1994; Duffy et al. 1996).

Spadro and Cullino (2005) concluded that the use of genetically modified

microorganisms could play an important role in crop production and protection.

Genetic manipulation could result in new biocontrol strains with increased produc-

tion of toxic compounds or lytic enzymes, improved space or nutrient competence,

wider host range or enhanced tolerance to abiotic stress (Glick and Bashan 1997).

Thus, biocontrol performance of soil pseudomonads may be improved by the

introduction of antibiotic biosynthetic genes (Maurhofer et al. 1992; Haas and

Keel 2003). Recombinant DNA strains with greatly increased diacetyl phloroglu-

cinol (DAPG) and phenazine-1-carboxamide (PCN) antibiotics production have

been constructed (Mavrodi et al. 1998, 2001). The production of DAPG and PCN

could be placed under the control of strong promoters or of exudate-induced or

rhizosphere-induced promoters (Mavrodi et al. 2006). Genes and enzymes involved

in the biocontrol mechanism could also be applied directly or transferred to crops.

From the perspective of developing nations, these are exciting strategies that

may help to increase yield while reducing the inputs and environmental problems.

However, most of the microbial biodiversity in soil remains unexplored and much

work remains to be done to first identify and then characterize microorganisms that

could be used in such applications. Furthermore, such approaches require a detailed

knowledge of the molecular signaling that takes place between plants and microbes

to drive expression of desirable traits and to suppress unwanted effects in a

controlled manner. Future strategies are required to clone genes involved in the

production of antibiotics, siderophores and other metabolites, and to transfer these

cloned genes into the rhizobacterial strains having good colonization potential

along with other beneficial characteristics such as N2 fixation, P-solubilization

and/or hormone production. Exploiting plants and microbes by using such an


integrated approach requires a coordinated strategy to understand the degree

and complexity of plant-microbe interactions employing modern “genomics/

proteomics” technologies. The generation of complete genomic sequences for

plant-associated bacteria, including pathogens and symbionts is already increasing

our knowledge of these organisms. The increasing amount of genomic data avail-

able for the model plant species and their associated microorganisms, will assist in

determining the most suitable beneficial bacterial strains for inoculation. In the

near future, the molecular tools adopted in manipulation of bacterial traits are

likely to improve the availability of nutrients, efficiency of phytoremediation and

enhancement of biocontrol activity that will consequently improve the crop pro-

ductivity and also protect the food chain by reducing levels of agrochemicals in

food crops.

9.5 Conclusion

Although striking advances have been made in understanding the molecular and

biochemical mechanism regulating N2 fixation, P solubilization and hormone

production, this has yet to be translated into applied environments. To overcome

the problem of establishment of inoculated microbes, the beneficial bacteria

intended for inoculation should be selected from local ecological niches and

reinoculated into the same environment to ensure the desired benefits. The effects

of soil and environmental factors on the physiology and ecology of introduced

microorganisms are still poorly understood. Research is therefore, needed to under-

stand the in situ physiology of inoculant cells and strategies must be developed as to

how such microbes could be manipulated for desired performance. For example,

the use of reporter genes inserted either randomly or directly into the bacterial

genome may allow the specific detection and possible enhancement of in situ gene

expression in inoculant cells.

The complex interactions among the PGPR, the pathogen, the plant and the

environment are responsible for the variability observed in disease suppression

and plant growth promotion. However, genetic manipulation of PGPR has the

potential to construct significantly better strains with improved biocontrol efficacy

(Trevors et al. 1990; Chet et al. 1993). Further, the efficacy of biocontrol bacteria

can be improved by developing better cultural practices and delivery systems that

favor their establishment in the rhizosphere. From the application point of view,

consortia of ecologically diverse strains for N2 fixation, P-solubilization, root growth

promotion among others, should be practiced instead of single strain. In near future,

both traditional and biotechnological approaches could be employed to increase

rates of N2 fixation, P solubilization, hormone production and increase in efficiency

of biocontrol activity along with bioremediation of contaminants, leading to

increase in crop yield under sustainable agricultural crop production system.


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Microbes for Legume Improvement || Growth Promotion of Legumes by Inoculation of Rhizosphere Bacteria